Strigolactone biosynthesis catalyzed by cytochrome P450 and sulfotransferase in sorghum.

Root parasitic plants such as Striga, Orobanche, and Phelipanche spp. cause serious damage to crop production world-wide. Deletion of the Low Germination Stimulant 1 (LGS1) gene gives a Striga-resistance trait in sorghum (Sorghum bicolor). The LGS1 gene encodes a sulfotransferase-like protein, but its function has not been elucidated. Since the profile of strigolactones (SLs) that induce seed germination in root parasitic plants is altered in the lgs1 mutant, LGS1 is thought to be an SL biosynthetic enzyme. In order to clarify the enzymatic function of LGS1, we looked for candidate SL substrates that accumulate in the lgs1 mutants and performed in vivo and in vitro metabolism experiments. We found the SL precursor 18-hydroxycarlactonoic acid (18-OH-CLA) is a substrate for LGS1. CYP711A cytochrome P450 enzymes (SbMAX1 proteins) in sorghum produce 18-OH-CLA. When LGS1 and SbMAX1 coding sequences were co-expressed in Nicotiana benthamiana with the upstream SL biosynthesis genes from sorghum, the canonical SLs 5-deoxystrigol and 4-deoxyorobanchol were produced. This finding showed that LGS1 in sorghum uses a sulfo group to catalyze leaving of a hydroxyl group and cyclization of 18-OH-CLA. A similar SL biosynthetic pathway has not been found in other plant species.

Shoot base responds to root-applied glutathione and functions as a critical region to inhibit cadmium translocation from the roots to shoots in oilseed rape (Brassica napus).

Glutathione (GSH) is a tripeptide involved in controlling heavy metal movement in plants. Our previous study showed that GSH, when site-specifically applied to plant roots, inhibits Cd translocation from the roots to shoots in hydroponically cultured oilseed rape (Brassica napus) plants. A factor that led to this inhibitory effect was the activation of Cd efflux from root cells. To further investigate the molecular mechanism triggered by root-applied GSH, Cd movement was non-invasively monitored using a positron-emitting tracer imaging system. The Cd absorption and efflux process in the roots were visualized successfully. The effects of GSH on Cd efflux from root cells were estimated by analyzing imaging data. Reanalysis of image data suggested that GSH applied to roots, at the shoot base, activated Cd return. Cutting the shoot base significantly inhibited Cd efflux from root cells. These experimental results demonstrate that the shoot base plays an important role in distributing Cd throughout the plant body. Furthermore, microarray analysis revealed that about 400 genes in the roots responded to root-applied GSH. Among these, there were genes for transporter proteins related to heavy metal movement in plants and proteins involved in the structure modification of cell walls.

Distinct deposition of ester-linked ferulic and p-coumaric acids to the cell wall of developing sorghum internodes.

Sorghum is important as a cereal crop, and also as livestock feed and a renewable energy crop because it produces a large amount of biomass. In grass plants like sorghum, hydroxycinnamates such as ferulic acids (FA) and p-coumaric acids (pCA) are characteristically ester-linked to the cell wall, and are believed to affect cell wall digestibility. Genetic manipulation of the esterification of FA and pCA to the cell wall appears to be one of the solutions to increase the digestibility of the cell wall so as to utilize sorghum biomass effectively. In this study, we measured esterified FA and pCA in each stage of internode elongation and determined the accumulation pattern of each hydroxycinnamate. The results revealed that FA were mainly accumulated during the cell elongation stage, and that pCA accumulation was increased after the cell elongation stage. Furthermore, 6 of the 12 sorghum BAHD acyltransferase genes were significantly expressed in the elongating internodes, suggesting that these genes might be involved in the feruloylation and/or p-coumaroylation of the cell wall in sorghum internodes.

Mutation of rice bc1 gene affects internode elongation and induces delayed cell wall deposition in developing internodes.

A rice COBRA-like gene, BRITTLE CULM1 (BC1) has been shown to be involved in assembling cell wall components and cellulose crystallinity, which determines mechanical strength in above ground organs. However, the detailed roles of BC1 in rice development are poorly understood. In this study, we found that, unlike the known brittle culm mutants, the internode length of the bc1 mutant was ~1.27 times longer than that of wild type in rice. In order to analyze the effects of bc1 mutation on internode development, we compared the deposition of cell wall components among each developmental stage of the elongating second internodes from wild type, Kinmaze, and the bc1 mutant. In wild type, histochemical observations of lignin revealed that lignin deposition was gradually increased after the cell elongation stage of the internodes. Cellulose and p-coumaric acid (pCA) content also gradually increased along with the progress of the developmental stage. The ferulic acid (FA) content rapidly increased in the cell elongation stage and decreased at the late secondary cell wall formation stage. In the bc1 mutant, the contents of cell wall components were lower than those of wild type from the cell elongation stage, in which the BC1 started to express at this stage in wild type. In the bc1 mutant, the deposition patterns of cell wall components, especially phenolic components including lignin, pCA, and FA, were delayed compared with those of wild type. These results suggest that the BC1 gene plays a role in synthesizing appropriate cell walls at each stage in the developing internode.

Effects of enhancing endogenous and exogenous glutathione in roots on cadmium movement in Arabidopsis thaliana.

Glutathione (GSH) is a thiol-containing compound involved in many aspects of plant metabolism. In the present study, we investigated how enhancing endogenous and exogenous GSH affects cadmium (Cd) movement and distribution in Arabidopsis plants cultured hydroponically. Transgenic Arabidopsis plants with a strong ability to synthesize GSH in roots were generated　by transforming the gene encoding the bifunctional γ-glutamylcysteine synthetase-glutathione synthetase enzyme from Streptococcus thermophiles (StGCS-GS). Enhancing endogenous and exogenous GSH decreased the Cd translocation ratio in different ways. Only exogenous GSH significantly inhibited Cd translocation from roots to shoots in wild-type and transgenic Arabidopsis plants. Our study demonstrated that GSH mainly functions outside root cells to inhibit Cd translocation from roots to shoots.

Ethylene biosynthesis controlled by NON-RIPENING: A regulatory conflict between wounding and ripening.

The phytohormone ethylene is involved in multiple aspects of morphological and physiological processes in plants. Tomato rapidly and transiently increases ethylene production during fruit ripening and in plant defense responses. The transcription factor non-ripening (NOR) has significant effects on fruit ripening via regulation of ethylene biosynthesis-related genes. The nor loss-of-function allele produces a basal level of ethylene during ripening, in contrast to the induced ethylene evolution observed upon Agrobacterium tumefaciens infection. The use of ACC deaminase represses ethylene production and significantly improves the efficiency of Agrobacterium-mediated T-DNA transfer in nor plants. Analyses of the transcription levels of the ethylene biosynthesis genes ACC synthase (ACS) and ACC oxidase (ACO) in nor plants revealed that the induced ethylene production was largely due to transcriptional accumulation of ACS2 and ACO1. Accumulation of ACS2 and ACO1 mRNA opposes NOR-mediated regulation in tomato fruit during ripening, and the feedback regulation of NOR is rendered ineffective by defense responses, thereby precluding the control of its own expression. The ethylene synthesis mechanisms respond properly to NOR-mediated transcriptional regulation that is differed through the wound-induced and ripening-induced signaling pathway.

Cloning and sequencing of the gene encoding the enzyme for the reductive cleavage of diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Geobacillus thermodenitrificans UZO 3.

We have previously reported that a cell-free extract prepared from Geobacillus thermodenitrificans UZO 3 reductively cleaves diaryl ether bonds of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), a dioxin with the highest toxicity, in a sequential fashion producing 3',4',4,5-tetrachloro-2-hydroxydiphenyl ether (TCDE) as the intermediate, and 3,4-dichlorophenol (DCP) as the final reaction product. The detection of TCDE implicated the discovery of an unprecedented dioxin-degrading enzyme that reductively cleaves the diaryl ether bonds. In this study, we report the cloning and sequencing of the dioxin reductive etherase gene dreE which codes for the 2,3,7,8-TCDD-degrading enzyme. We showed that dreE was expressed in Escherichia coli and that the product of the expression could reductively cleave diaryl ether bonds of 2,3,7,8-TCDD to produce TCDE. Furthermore, we established that the amino acid sequence encoded by dreE was homologous to an enzyme with yet unknown function that is encoded by a gene located in the riboflavin (vitamin B2) biosynthesis operon in Bacillus subtilis. We also showed that the amino acid sequence possesses a coenzyme A (CoA) binding site that is conserved in the N-acyltransferase superfamily. For the first time, the degradation of 2,3,7,8-TCDD at the molecular level using a enzyme of bacterial origin has been demonstrated. A novel mechanism model for the reductive cleavage of diaryl ether bond of 2,3,7,8-TCDD was also proposed.

Development of a stable Agrobacterium-mediated transformation protocol for Sorghum bicolor Tx430.

Sorghum is a recalcitrant crop for Agrobacterium-mediated genetic transformation. Several parameters related to Agrobacterium-mediated transformation were tested to optimize sorghum transformation frequencies. In this study, we evaluated pretreatment of sorghum variety Tx430 immature embryos using Agrobacterium strain GV2260. Pretreatment of immature embryos with heat (43°C) treatment for 15 or 21 min, and centrifugation resulted in a transformation efficiency of up to 1.9% of immature embryos treated. Although further optimization to enhance transformation efficiency is required, this study contributes to the genetic validation of genes of interest and molecular breeding in sorghum plants.

SMALL ORGAN SIZE 1 and SMALL ORGAN SIZE 2/DWARF AND LOW-TILLERING Form a Complex to Integrate Auxin and Brassinosteroid Signaling in Rice.

Although auxin and brassinosteroid (BR) synergistically control various plant responses, the molecular mechanism underlying the auxin-BR crosstalk is not well understood. We previously identified SMOS1, an auxin-regulated APETALA2-type transcription factor, as the causal gene of the small organ size 1 (smos1) mutant that is characterized by a decreased final size of various organs in rice. In this study, we identified another smos mutant, smos2, which shows the phenotype indistinguishable from smos1. SMOS2 was identical to the previously reported DWARF AND LOW-TILLERING (DLT), which encodes a GRAS protein involved in BR signaling. SMOS1 and SMOS2/DLT physically interact to cooperatively enhance transcriptional transactivation activity in yeast and in rice nuclei. Consistently, the expression of OsPHI-1, a direct target of SMOS1, is upregulated only when SMOS1 and SMOS2/DLT proteins are both present in rice cells. Taken together, our results suggest that SMOS1 and SMOS2/DLT form a keystone complex on auxin-BR signaling crosstalk in rice.

Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

Beta-ketoadipic acid and muconolactone production from a lignin-related aromatic compound through the protocatechuate 3,4-metabolic pathway.

In this work, the effects of PcaJ (beta-ketoadipate:succinyl-coenzyme A transferase)- and PcaD (beta-ketoadipate enol-lactone hydrolase)-inactivation on protocatechuic acid metabolism in Pseudomonas putida KT2440 were evaluated. Beta-ketoadipic acid was produced from protocatechuic acid by the inactivation of PcaJ as expected; however, a portion of the produced beta-ketoadipic acid was converted to levulinic acid through a purification step consisting of extraction from the culture and recrystallization. On the other hand, muconolactone was purified from the culture of the PcaD-inactivated mutant of KT2440, although beta-ketoadipate enol-lactone was supposed to be produced because it is the substrate of PcaD. Under aerobic conditions, it has been reported that lignin-related aromatics are metabolized through PCA 2,3- or 3,4- or 4,5-ring cleavage pathways, and muconolactone is an intermediate observed in the metabolism of catechol, not protocatechuic acid. Our results will provide a prospective route to produce muconolactone with a high yield through the protocatechuate-3,4-metabolic pathway.

Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD, the Cα-dehydrogenase from Sphingobium sp. strain SYK-6.

Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram-negative bacterium Sphingobium sp. SYK-6 possesses a Cα-dehydrogenase (LigD) enzyme that has been shown to oxidize the α-hydroxy functionalities in ß-O-4-linked dimers into α-keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of ß-O-4-linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol-ß-coniferyl alcohol ether and syringylglycerol-ß-sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon-optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC-MS/MS-based metabolite profiling indicated that levels of oxidized guaiacyl (G) ß-O-4-coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1- to 2.8-fold increased level of G-type α-keto-ß-O-4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana.

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4'-feruloyl ethers. In-depth quantitative analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following ß-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.

A novel AP2-type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway.

The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants.

DWARF50 (D50), a rice (Oryza sativa L.) gene encoding inositol polyphosphate 5-phosphatase, is required for proper development of intercalary meristem.

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.